Project Summary Proliferating cell nuclear antigen (PCNA) is an essential component of the replisome, and it enhances the processivity of the DNA polymerases in DNA synthesis. In addition, in response to DNA damage, PCNA is ubiquitinated at lysine 164 (K164) to bypass DNA lesions. In unperturbed cells, the same K164 residue can also be conjugated with either SUMO1 or SUMO2, and SUMO1-PCNA has been implicated in recruiting PARI helicase to suppress homologous recombination. However, the source of replication obstacle that triggers PCNA SUMOylation is yet to be defined, and the regulators of PCNA SUMOylation are not known. It is also not clear if SUMO2-PCNA functions redundantly to SUMO1-PCNA. Our newly published data argue that human SUMO2-PCNA has a unique function in transcription that is not shared by SUMO1-PCNA, because only SUMO2-PCNA is associated with transcriptionally active chromatin. Even though PCNA SUMO2 conjugation occurs in S-phase, SUMO2-PCNA is induced by RNA polymerase II - dependent transcription and requires the RNA polymerase II - interacting protein, RECQ5 DNA helicase. Importantly, cells with reduced SUMO2-PCNA accumulate transcription-induced DNA double-strand breaks during S-phase. Therefore, our data support a conceptually innovative model for a role of SUMO2-PCNA in resolving transcription-replication conflicts to minimize genomic instability. The goal of this proposal is (1) to identify the molecular factors that facilitate the transcription-induced SUMO2 conjugation of PCNA, (2) to identify the molecular mechanism by which SUMO2- PCNA resolves transcription-replication conflicts, and (3) to elucidate the contribution of SUMO2-PCNA toward preventing genomic instability and neoplastic transformation, as transcription-replication conflicts are major source for common fragile site instability.